Human Neuropathy Target Esterase Catalyzes Hydrolysis of Membrane Lipids*

A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues inDrosophila and yeast and is detected in vitroby assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727–1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A2(iPLA2) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA2 inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (V max ∼0.01 μmol/min/mg and K m ∼0.4 mm for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of 14C-labeled products from two differentially labeled 14C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (V max ∼20 μmol/min/mg andK m ∼0.05 mm for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (V max ∼1 μmol/min/mg andK m ∼0.4 mm for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di- or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking.

Neuropathy target esterase (NTE) 1 is an integral membrane protein in neurons and some non-neural cell types; it was originally identified as the primary site of action for those organophosphates which, in humans and other vertebrates, cause a syndrome characterized by axonal degeneration (1). The NTE homologue in Drosophila, the swiss cheese protein, is essential for fly brain development (2). In the mouse, NTE is present in neurons from their earliest appearance in the nervous system and so is well placed to play a similar role in mammalian neural development (3). NTE also has a homologue in yeast (4), suggesting that it has functions beyond the nervous system and mediates a biochemical reaction highly conserved through evolution.
In keeping with its reactivity with organophosphates, NTE belongs to the serine hydrolase class of enzymes. Since its discovery more than 30 years ago (5), NTE has been detected in vitro by assays with non-physiological ester substrates, most commonly with phenyl valerate (6). Clues to the cellular functions of NTE might be provided by identifying its natural substrate. Data on this point are sparse, but using artificial substrates, NTE activity in brain homogenates has been shown to catalyze hydrolysis of ester rather than peptide or amide bonds (7,8). In addition, inhibition of the phenyl valerate hydrolase activity of NTE by a homologous series of alkyl saligenin cyclic phosphonates (9) and alkyl-thiotrifluoromethyl ketones (10) suggests that the active site of the enzyme has a preference for esters of carboxylic acids with an alkyl chain length of at least 9 -10 carbon atoms (1). This hints that the natural substrate of NTE may be an ester of a fatty acid, such as a phospholipid or an acylglycerol.
Human NTE is a polypeptide of 1327 residues and has two major domains (4) as follows: a C-terminal catalytic domain containing the active site serine residue (Ser-966) that reacts with organophosphates and phenyl valerate; and an N-terminal putative regulatory domain that contains sequences similar to cyclic AMP-binding proteins. We have expressed in Escherichia coli a recombinant protein (corresponding to NTE residues 727-1216) called NEST which composes the esterase domain of NTE (11). NEST is very hydrophobic, and the purified protein must be incorporated into phospholipid liposomes to acquire a conformation with full phenyl valerate hydrolase activity (12). In this respect, NEST appears similar to enzymes such as cytoplasmic phospholipase A 2 (13) and triacylglycerol lipase (14) which show the phenomenon of interfacial activation, i.e. they exhibit full catalytic activity only when at a lipid-water interface. Here, we use NEST to investigate the possibility that NTE has lipase-or phospholipase-A-like activity with membrane-associated lipids as its potential substrates.
All other lipid substrates, putative lipase inhibitors, and bee venom secretory phospholipase A 2 were purchased from Sigma. Lipid substrates were prepared by evaporating chloroform solutions under a stream of nitrogen gas and then resuspending the residue in PEN buffer (50 mM sodium phosphate, 0.5 mM EDTA, 300 mM NaCl, pH 7.8) by sonication (MSE Sanyo Soniprep 150 probe sonicator) at maximum power for 10 min to give stock suspensions at a concentration of 5 mM. Free fatty acid assay kit (half-micro test) was from Roche Diagnostics. The sources of all other materials have been described previously (11).
Expression and Isolation of NEST-NEST (human NTE residues 727-1216 with N-terminal T7 and C-terminal His 6 tags) was expressed in E. coli BL21 pLysS, extracted, and isolated by nickel chelate and gel filtration chromatography in solutions containing CHAPS as described previously (11). NEST, with its catalytic serine residue mutated to alanine (11), was expressed and isolated in identical fashion. Before assaying its phenyl valerate hydrolase activity, purified NEST was diluted with 0.3% CHAPS to a protein concentration of 0.1 mg/ml, mixed with dioleoylphosphatidylcholine (DOPC; 10 mg/ml in 10% (w/v) CHAPS) in a ratio of 1:4 (w/w), and then dialyzed overnight at room temperature against PEN buffer.
Enzyme Assays-Phenyl valerate hydrolase activity was determined by the method of Johnson (6) as described previously (11). Purified NEST-DOPC complexes were diluted in 50 mM Tris-HCl, 1 mM EDTA to a protein concentration of 20 -80 ng/ml and then incubated for 20 min at 37°C with inhibitors before addition of phenyl valerate in Triton X-100 (final concentrations of 1.4 and 0.24 mM, respectively) and incubation for a further 20 min. NEST lipase activity was assayed by determining the liberation of free fatty acid from various lipid substrates. For lysophospholipids and monoacylglycerols, incubations contained 2.5 mM substrate, NEST  For more detailed evaluation of phospholipase A 2 activity, 14 C-labeled phospholipids (ϳ0.4 ϫ 10 6 dpm) were incubated at 37°C with NEST (8 -20 g) in 0.05 ml of PEN buffer containing 2-4 mM CHAPS. Reactions were stopped by the addition of 0.1 ml of chloroform/methanol/acetic acid (2:1:0.02), and after vortexing and centrifugation, the organic phase solvent was evaporated, and the residue was redissolved in ethanol containing oleic acid (20 mg/ml). Aliquots were subjected to thin layer chromatography on Silica G plates run in hexane/ether/acetic acid (80:20:1). In this system, fatty acids are well resolved from phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) which remain very close to the origin. Spots containing either phospholipid or fatty acid were scraped into scintillation vials, and radioactivity was determined by scintillation counting (16). To resolve [ 14 C]LPC, samples (mixed with unlabeled 1-oleoyl-LPC) were run on Silica G plates in chloroform/methanol/formic acid/water (60:30:8:2) (17). In this system, LPC has an R F ϳ0.5, whereas PC and fatty acids co-migrate near the solvent front.

NEST and Calcium-independent Phospholipase A 2 , Similarities in Active Site Sequence and Inhibitor Sensitivities of Phenyl Valerate
Hydrolase Activity-A BLAST search identified significant sequence similarity (28% identity) between an ϳ160-residue region of NEST and calcium-independent phospholipase A 2 (iPLA 2 ; Fig. 1). Among key conserved residues in both proteins were those corresponding to the active site serine (Ser-966) and two aspartates (Asp-960 and Asp-1086) shown previously to be essential for the phenyl valerate hydrolase activity of NEST (11). Outside this region there was no homology between NTE and iPLA 2 (Fig. 1).
Inhibition of the phenyl valerate hydrolase activity of NEST-DOPC preparations by fatty acid TFMKs led us to ask whether fatty acids themselves would be inhibitory. Preincubating NEST-DOPC with 50 M oleic acid abolished phenyl valerate hydrolase activity, whereas palmitic and arachidonic acids at this concentration had marginal (25% inhibition) or no effect, respectively (Fig. 2a). Similarly, palmitate and arachidonate at concentrations of 30 M did not inhibit iPLA 2 , although this study did not report the effect of oleic acid (18).
As free fatty acid and lysophospholipid are the products of PLA 2 activity, we examined possible effects of lysophospholip- ids on NEST-DOPC phenyl valerate hydrolase activity by preincubating with the enzyme before addition of substrate: 1-palmitoyl-LPC was inhibitory with an IC 50 value of ϳ15 M, and an alkyl ether analogue, 1-O-hexadecyl-LPC, was even more potent with an IC 50 Ͻ 7.5 M (Fig. 2b). By contrast, 1-O-hexadecyl, 2-arachidonoyl-PC had only a marginal effect (Fig. 2b).
To investigate the nature of the inhibition of phenyl valerate hydrolase activity of NEST-DOPC by oleic acid and lysophospholipids, we directly added these inhibitors to assays in the presence of varying concentrations of phenyl valerate substrate (Fig. 3). Oleic acid was much less potent as an inhibitor when added direct to the assay (Fig. 3a) than when preincubated with the enzyme before addition of substrate (cf. Fig. 2a). For example, in the presence of the standard concentration of phenyl valerate (1.4 mM), 50 M oleic acid caused only ϳ15% inhibition, increasing to ϳ45% inhibition in the presence of 0.35 mM phenyl valerate (Fig. 3a). By contrast, LPC was almost as potent when added direct to the assay as when preincubated and at 25 M caused 50% inhibition in the presence of 1.4 mM phenyl valerate (Fig. 3b). Lineweaver-Burk (1/v versus 1/s) transformations (not shown) of the data of the type in Fig. 3 were readily fit to straight lines allowing determination of kinetic values for phenyl valerate substrate (V max ϭ 4.82 Ϯ 0.52 mmol/min/mg; K m ϭ 0.76 Ϯ 0.09 mM; n ϭ 5). However, in the presence of either oleic acid or LPC, Lineweaver-Burk plots intersected the y axis at a lower value than in the absence of these inhibitors, suggesting an increase in V max . It is notable that preincubation of NEST-DOPC with 10 M fatty acid caused a small (20%) increase in phenyl valerate hydrolase activity (Fig. 2a). Thus, the interactions between NEST-DOPC, CHAPS, phenyl valerate, and either oleic acid or LPC are complex and cannot be described kinetically in terms of pure competitive or non-competitive inhibition.
NEST Slowly Liberates Free Fatty Acid from Phospholipids-NEST is extracted and isolated from bacterial lysates in solutions containing CHAPS, and under these conditions, its phenyl valerate hydrolase activity is lost, partly because this activity requires association with phospholipid and partly because concentrations of CHAPS greater than its critical micellar concentration (4 -8 mM) are inhibitory (12). Restoration of We established conditions (2.5 mM phospholipid, 3.2 mM CHAPS) for assaying the lipase activity of NEST as described under "Experimental Procedures." After 1 h at 37°C, ϳ0.8 mol of oleic acid was liberated from DOPC per mg of NEST (Fig. 4a). This slow rate of hydrolysis was not substantially altered by several changes to the incubation conditions as follows: varying CHAPS concentration between 1.6 and 8 mM; incorporating DOPC into mixed lipid vesicles (various ratios of DOPC/dioleoylethanolamine/sphingomyelin/cholesterol); by adding calcium or magnesium ions; or by adding ATP, which has been reported to enhance iPLA 2 activity (20) (data not shown).
Whereas 1-O-hexadecadyl, 2-arachidonoyl-PC is a substrate for iPLA 2 (20), it was not detectably hydrolyzed by NEST (Fig.  4b). This raised the possibility that NEST mediates selective cleavage of the sn-1 bond of phospholipids; to examine this issue we incubated NEST with differentially labeled [ 14 C]phosphatidylcholines (Table I). Approximately twice as much 14 (Table I). These results strongly suggest that NEST mediates a very slow cleavage of the sn-2 bond of phospholipid followed by a rapid hydrolysis of the resulting lysophospholipid product, possibly before this product is released from the enzyme. Cleavage of the sn-2 bond of 1-O-hexadecyl, 2-arachidonoyl-PC by NEST would yield a non-hydrolyzable lysophospholipid product; the latter may dissociate only very slowly from the enzyme and hence inhibit binding of further phospholipid substrate, giving rise to the result observed in Fig. 4b.
NEST liberated [ 14 C]oleic acid from sn-2-labeled PC at rates that were approximately linear over a period of 15 min (Fig.   FIG. 6. NEST potently catalyzes lysophospholipid hydrolysis and does not require additional phospholipid. a, NEST was incubated with 1-palmitoyl-LPC or 1-oleoyl-LPC, and reactions were stopped at the indicated times with MAFP and free fatty acid determined as under "Experimental Procedures." b, NEST that had been incorporated into DOPC liposomes by overnight dialysis (see "Experimental Procedures") or NEST alone (both at 0.1 g of protein/assay) was incubated with 1-palmitoyl-LPC, and free fatty acid was liberated determined as in a. 5a). Thereafter, 15-min assays were used to determine the dependence of reaction rate on substrate concentration. Lineweaver-Burk transformations of these data (Fig. 5b) allowed determination of values for 1-palmitoyl, 2-oleoyl-PC of V max ϭ 11.6 Ϯ 1.1 nmol/min/mg and K m ϭ 0.37 Ϯ 0.12 mM (from three experiments).
Lysophospholipids Are the Most Avidly Hydrolyzed Lipid Substrate for NEST-Fatty acid was generated rapidly when NEST was incubated with LPC. With 1-palmitoyl-LPC, initial rates of ϳ20 mol/min/mg were about twice those observed with 1-oleoyl-LPC (Fig. 6a) and were ϳ200 times faster than those with 1-palmitoyl, 2-arachidonoyl-PC (cf. Fig. 4b). As with the diacylphospholipid substrates, rates declined after 2-5 min, and this was not due to substrate depletion.
Among the lipid substrates tested with NEST, lysophospholipids gave by far the fastest initial rates. The affinity of the active site of NEST for lysophospholipids may underlie the potent inhibition by these compounds and their ether analogues of phenyl valerate hydrolase activity (cf. Figs. 2b and  3b). However, unlike phenyl valerate hydrolase activity, NEST lipase activity toward lysophospholipid (Fig. 6b) and other lipid substrates did not require incorporation into DOPC vesicles to achieve interfacial activation, suggesting that in these cases the substrate itself fulfills this requirement.
We established conditions under which linear rates of hydrolysis of 1-palmitoyl-LPC could be observed (Fig. 7a). Lineweaver-Burk transformation of data obtained under these conditions (Fig. 7b) allowed the determination of values for V max ϭ 20.8 Ϯ 2.5 mol/min/mg and K m ϭ 0.054 Ϯ 0.006 mM (from four experiments).
Other Naturally Occurring Lipid Substrates for NEST-NEST hydrolyzed monoacylglycerols, with a marked preference for the sn-1 isomer. After a 5-min incubation, 8 -10 times more fatty acid was liberated from 1-palmitoylglycerol than from the 2-isomer (Fig. 8a). Although the first 2-5 min of NEST-catalyzed hydrolysis of 1-and 2-oleoylglycerol and 2-arachidonylglycerol were at least as rapid as for 2-palmitoylglycerol, the rate diminished dramatically after this time (Fig.  8b). Thus, with certain members of each class of lipid substrate (phospholipids, lysophospholipids, and acylglycerols), NESTcatalyzed release of fatty acid shows an initial burst of variable duration and then slows markedly. A similar, poorly understood stalling in reaction rate has been reported for several phospholipases (21)(22)(23). Nevertheless, we were able to establish conditions under which approximately linear rates of hydrolysis of 1-palmitoylglycerol could be measured (Fig. 9a). Lineweaver-Burk transformations of these rates (Fig. 9b) allowed determination of values for V max ϭ 1.37 Ϯ 0.47 mol/min/mg and K m ϭ 0.37 Ϯ 0.09 (from three experiments).
In contrast to the relatively brisk hydrolysis of monoacylglycerols, NEST did not catalyze detectable fatty acid release from di-or triacylglycerols nor from a variety of acyl amides including sphingomyelin, anandamide, and N-oleoylethanolamine (data not shown). Free fatty acid was slowly liberated when a suspension of cholesteryl oleate was incubated with NEST, at rates similar to those with DOPC (data not shown).

DISCUSSION
We have shown here that NEST, the recombinant esterase domain of NTE, liberates fatty acid from phospholipids, monoacylglycerols, and lysophospholipids. Thus, more than 30 years after the discovery of NTE, we have identified naturally occurring, membrane-associated lipids as its potential endogenous substrates. This demonstration that NTE is a putative lipase provides new clues to its possible cellular functions.
Under the experimental conditions in this report, lysophospholipids were by far the most avidly hydrolyzed substrate for NEST. It has been stressed repeatedly that the rates and selectivities of bond cleavage observed in lipase assays in vitro are profoundly affected by the physicochemical nature of the substrate (14,16,20,23). Nevertheless, it seems reasonable to consider the possibility that lysophospholipids may be a physiological substrate for NTE.
The presence of a homologue of NTE in yeast (4) suggests that this enzyme mediates a cellular process conserved throughout much of eukaryotic evolution. NTE itself is firmly associated with intracellular membranes, and from a combination of esterase assays on brain subcellular fractions (30), immunohistochemistry on brain sections (31), and distribution of green fluorescent protein-tagged NTE in transfected COS-7 cells, 2 we have tentatively concluded that NTE may be localized predominantly in the endoplasmic reticulum and Golgi complex. What might be the biological significance of NTEmediated lysophospholipase activity in the endoplasmic reticulum/Golgi of various cells from yeast to neurons?
There is a growing recognition that, along with other factors, the relative concentrations of diacyl-and lysophospholipids contribute to the degree of curvature of biological membranes and that this is an important determinant in the process of membrane fission, tubulation, and fusion (32)(33)(34). Addition to cultured mammalian cells of various PLA 2 inhibitors, including bromoenol lactone and arachidonoyl-TFMK, causes reversible fragmentation of the Golgi complex (35,36). It has been suggested that a PLA 2 activity may be required for maintenance of Golgi complex architecture (35); this would result in continuous production of lysophospholipid within the Golgi. Indeed, in Golgi membranes (of rat kidney and liver), lysophospholipids compose 3-4% of the total phospholipid (37). It has been dem-onstrated that CtBP/BARS-catalyzed acylation of lysophosphatidic acid to form phosphatidic acid induces fission of Golgi membranes (33). Another mechanism for acutely reducing the steady-state lysophospholipid level might involve regulated lysophospholipase activity. If ligand binding to the N-terminal domain of the NTE, which has sequence similarity to cyclic AMP-binding proteins (4), modulates the lipase activity of NTE toward its natural substrate, then this might allow inducible alteration of Golgi architecture and contribute to processes such as regulated protein secretion.